SUMMARY: The generation of induced pluripotent stem cells (iPSCs) is extremely inefficient, suggesting that transcription factors have to overcome somatic barriers that resist cell fate change. Dissecting the mechanisms that resist the reprogramming of somatic cells into iPSCs is a common goal to this P01 group and is expected to provide valuable insights into the mechanisms that normally maintain cellular identity and prevent disease. In order to uncover novel barriers to reprogramming, we performed an unbiased shRNA enrichment screen during iPSC formation. This led to the identification of the RNA processing factor Nudt21 and the protein modifier Sumo2 as the most prominent hits, increasing iPSC formation by orders of magnitude. Nudt21 has recently been implicated in the regulation of alternative polyadenylation (APA) patterns, whereas Sumo2 is a key effector of the sumoylation pathway that influences protein function. Although both Nudt21 and Sumo2 are involved in crucial post-transcriptional processes affecting the expression of dozens of mRNAs or proteins, little is currently known about their targets and the mechanisms by which they control cell fate. In addition, both the human SUMO-conjugating enzyme UBE2i and NUDT21 were positive hits in Zaret's proteomic analysis of heterochromatin and will be investigated in that context for function and associated subcomplex identity. Along these lines, chromatin-associated sumoylation has been causally linked to epigenetic gene silencing while APA patterns are thought to be influenced by preexisting chromatin structure, thus providing intriguing connections to iPSC generation, which involves major rearrangements of chromatin landscapes. Here, we outline three complementary aims to dissect the cellular, molecular and functional roles Nudt21 and Sumo2 play in cellular reprogramming and pluripotency. In the first aim, we will explore the cellular contexts in which Sumo2 and Nudt21 suppression promotes iPSC formation. We will determine whether Nudt21 and Sumo2 depletion enhances reprogramming of different mouse and human cell types into iPSCs, whether other components of the sumoylation and APA pathways phenocopy these effects and which stages of reprogramming are affected by these perturbations. In the second aim, we will elucidate the molecular mechanisms by which Nudt21 and Sumo2 resist reprogramming. We will utilize unbiased approaches to identify targets of Sumo2 and Nudt21, test their function during reprogramming and study their roles in heterochromatin dynamics with the Zaret lab. In addition, we will dissect the links between sumoylation, APA, OKSM binding and chromatin structure by working with the Plath lab. In the third aim, we will examine whether the functions of Nudt21 and Sumo2 are conserved across related cell fate transitions, including exit from and re-entry into nave pluripotency. Collectively, this P01 application will contribute to our understanding of how sumoylation and alternative polyadenylation control cell fate change in different cellular contexts by using Nudt21 and Sumo2 as tools.